This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The apparent ability of M. tuberculosis to become dormant in the human host, leading to asymptomatic latent infection, has stimulated great interest in understanding how cell growth and cell division are regulated in this organism. A longstanding observation that "spent" or "conditioned" medium from bacterial cultures is able to stimulate growth of dormant cells, led to the identification of a resuscitation promoting factor (Rpf) by purifying from spent medium a component that was able to stimulate growth in the actinomycete Micrococus luteus. Rpf is small protein that has homologues in other actinobacteria, including M. tuberculosis, which has five rpf genes. Functional studies of these genes in M. tuberculosis has shown that individually they are not required for resuscitation of dormant Mtb cells and single rpf mutant strains do not have other growth or morphologic phenotypes. When two or more rpf genes are inactivated, however, growth or resuscitation defects are observed. The recent demonstration that the Rpf's are PGN hydrolases suggests that growth stimulation of dormant cells may result from the enzymatic activity of these secreted proteins, possibly through alterations in PGN structure or through the interaction of PGN degradation products with the bacterial cell surface. A domain found to occur in the extracellular portions of penicillin binding proteins and serine/threonine kinases (PASTA domain) was identified by bioinformatic analysis and predicted to bind to the stem peptide of un-crosslinked PGN precursors based on the structure of PBP2X of Streptococcus pneumoniae bound to a cephalosporin antibiotic. Recently the PASTA domain of a Ser/Thr kinase of B. subtilis was shown to bind both intact and hydrolyzed PGN. Incubation of B. subtilis spores with PGN stimulated spore germination and increased Ser/Thr phosphorylation. Some specificity with respect to the source of PGN and these functional effects was observed, suggesting a preference for meso-diaminopimelic acid (m-DAP)-containing PGN in stimulating spore germination. The M. tuberculosis genome encodes two proteins that contain PASTA domains, the Ser/Thr protein kinase PknB (Rv0014c) whose extracellular region comprises four PASTA domains, and the bifunctional penicillin binding protein PBP2 (PonA2, Rv3682), which has a single PASTA domain at the extreme carboxy-terminus of protein distal to the extracellular transpeptidase and transglycosylase-containing domains. In this work we determined that PknB is localized to septum and cell poles.